Polymer supports for nucleic acid synthesis

ABSTRACT

The invention provides passivated organic polymer supports, processes for their preparation and processes for their use in oligonucleotide synthesis that allow for highly efficient solid phase synthesis of oligonucleotides.

This application is a divisional of Ser. No. 08/562,841; filed Nov. 27,1995 now U.S. Pat. No. 5,668,268.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the chemical synthesis of oligonucleotides andto materials and processes that are useful in such synthesis.

2. Summary of the Related Art

Oligonucleotides have become indispensable tools in modern molecularbiology, being used in a wide variety of techniques, ranging fromdiagnostic probing methods to PCR to antisense inhibition of geneexpression. This widespread use of oligonucleotides has led to anincreasing demand for rapid, inexpensive and efficient methods forsynthesizing oligonucleotides.

The synthesis of oligonucleotides for antisense and diagnosticapplications can now be routinely accomplished. See e.g., Methods inMolecular Biology, Vol 20: Protocols for Oligonucleotides and Analogspp. 165-189 (S. Agrawal, Ed., Humana Press, 1993).; Oligonucleotides andAnalogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991);and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech.6: 12 (1995); and Anti-sense Research and Applications (Crooke andLebleu; Eds., CRC Press, Boca Raton, 1993). Early synthetic approachesincluded phosphodiester and phosphotriester chemistries. Khorana et al.,J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry foroligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179(1978), discloses phosphotriester chemistry for synthesis ofoligonucleotides and polynucleotides. These early approaches havelargely given way to the more efficient phosphoramidite andH-phosphonate approaches to synthesis. Beaucage and Caruthers,Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use ofdeoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawaland Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimizedsynthesis of oligonucleotides by the H-phosphonate approach.

Both of these modern approaches have been used to synthesizeoligonucleotides having a variety of modified internucleotide linkages.Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teachessynthesis of oligonucleotide methylphosphonates using phosphoramiditechemistry. Connolly et al., Biochemistry 23: 3443 (1984), disclosessynthesis of oligonucleotide phosphorothioates using phosphoramiditechemistry. Jager el al., Biochemistry 27: 7237 (1988), disclosessynthesis of oligonucleotide phosphoramidates using phosphoramiditechemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083(1988), discloses synthesis of oligonucleotide phosphoramidates andphosphorothioates using H-phosphonate chemistry.

Solid phase synthesis of oligonucleotides by any of the known approachesordinarily involves the same generalized protocol. Briefly, thisapproach comprises anchoring the 3′-most nucleoside to a solid supportfunctionalized with amino and/or hydroxyl moieties and subsequentlyadding the additional nucleosides in step-wise fashion. Desiredinternucleoside linkages are formed between the 3′ functional group(e.g., phosphoramidite group) of the incoming nucleoside and the 5′hydroxyl group of the 5′-most nucleoside of the nascent, support-boundoligonucleotide.

Refinement of methodologies is still required, however, particularlywhen making a transition to large-scale synthesis (10 umol to 1 mmol andhigher). See Padmapriya et al., Antisense Res. Dev. 4: 185 (1994).Several modifications of the standard phosphoramidite methods havealready been reported to facilitate the synthesis and isolation ofoligonucleotides. See e.g., Padmapriya et al., supra; Ravikumar et al.,Tetrahedron 50: 9255 (1994); Theisen et al., Nucleosides & Nucleotides12: 43 (1994); and Iyer et al., Nucleosides & Nucleotides 14: 1349(1995) (Kuijpers et al., Nucl. Acids Res. 18: 5197 (1990); and Reddy etal., Tetrahedron Lett. 35: 4311 (1994).

One limitation in solid phase synthesis resides in the nature of thesolid phase support upon which the oligonucleotide is synthesized. Avariety of solid support materials have been described for solid phaseoligonucleotide synthesis, the most prevalent of which iscontrolled-pore glass (CPG). (See, e.g., Pon, Methods in Molec. Biol.20: 465 (1993)). Unfortunately, CPG suffers certain limitations thatprevent it from being an ideal support material. See e.g., Ron et al.,Biotechniques 6: 768 (1988); McCollum et al., Nucleosides andNucleotides 6: 821 (1987); Bardella et al., Tetrahedron Lett. 31:6231-6234 (1990) For example, CPG is unstable under the standardammonium hydroxide procedure that is used to deprotect theoligonucleotide and to cleave it from the solid support. In addition,oligonucleotide synthesis using CPG as the solid support results inrather high levels of n-1 contaminant in the synthesis product.

To overcome these problems, various attempts have been made to developpolymer supports to replace CPG. See e.g., Gao et al., Tetrahedron Lett.32: 5477-5479 (1991); The Gene Assembler™, A Fully Automated DNASynthesizer, Pharmacia Fine Chemicals, Uppsala, Sweden (1986). The useof organic supports in this context has been explored. Reddy et al.,Tetrahedron Lett. 35: 5771-5774 (1994) discloses an organic supportbased on native Fractogel (“Toyopearl”, TosoHaas, Philadelphia, Pa.).Fractogel, however, has inherent limitations as a support foroligonucleotide synthesis, due to its low density when packed inacetonitrile and its limited pore volume per unit bed volume. Althoughit would be desirable to replace CPG with a support that lacks itslimitations, none of the polymer supports developed to date haveprovided the efficiency that CPG provides.

There is, therefore, a need for polymer supports for oligonucleotidesynthesis that provide the efficiency of CPG, but that do not sufferfrom the instability or n-1 contamination problems inherent in CPG.

BRIEF SUMMARY OF THE INVENTION

The invention provides passivated organic polymer supports, processesfor their preparation and processes for their use in oligonucleotidesynthesis that allow for highly efficient solid phase synthesis ofoligonucleotides. The efficiency of synthesis provided when using theorganic polymer supports according to the invention is at least as goodas that provided by controlled pore glass (CPG). Unlike CPG, the organicpolymer supports according to the invention are highly stable understandard ammonium hydroxide conditions used to deprotect theoligonucleotides and to cleave them from the solid support. In addition,solid phase oligonucleotide synthesis using the organic polymer supportsaccording to the invention results in greatly reduced production of n-1contaminant oligonucleotide.

In a first aspect, the invention provides a passivated organic polymersupport for solid phase synthesis of oligonucleotides. The passivatedorganic polymer support according to the invention comprises a pluralityof microscopic particles. Each particle has amino and/or hydroxyl groupscovalently bound to the particle. Each particle further has nucleosidescovalently bound to some of the amino and/or hydroxyl groups. At leastsome of the amino and/or hydroxyl groups that are not covalently boundto nucleosides are covalently bound to hydrophobic passivating groups.

In a second aspect, the invention provides a process for passivating anorganic polymer support for oligonucleotide synthesis. The processaccording to the invention comprises introducing hydrophobic passivatinggroups at the site of free amino and/or hydroxyl groups that arecovalently bound to the particles that comprise the organic polymersupport. Organic polymer supports for oligonucleotide synthesis haveamino and/or hydroxyl groups covalently bound to the particles thatcomprise the support. Some of the amino and/or hydroxyl groups arecovalently bound to nucleosides, while others remain as free aminoand/or hydroxyl groups. The presence of these amino and/or hydroxylgroups lends a hydrophilic character to the particles. In the processaccording to the invention, the hydrophilic character of the particlesis reduced by covalently attaching hydrophobic passivating groups to theamino and/or hydroxyl groups. Passivation of the particles in thismanner results in greatly improved efficiency of oligonucleotidesynthesis.

In a third aspect, the invention provides an improved process for solidphase oligonucleotide synthesis. In this improved process according tothe invention, the improvement comprises carrying out solid phasesynthesis on the passivated organic polymer support according to theinvention. This process of oligonucleotide synthesis according to theinvention produces oligonucleotides at least as efficiently as processesutilizing CPG, but with greatly reduced contamination by n-1 and withoutchemical breakdown of the solid support.

The organic polymer supports and process for their use according to theinvention are useful for synthesizing oligonucleotides on a scaleranging from small laboratory scale to large commercial scale. Thus, theorganic polymer supports and process for their use according to theinvention can be used to supply oligonucleotides for research purposes,for diagnostic purposes and for therapeutic purposes using the antisenseapproach.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a surface area within a pore of a particle that comprisesan organic polymer support according to the invention.

Panel A shows the surface before addition of the nucleosides.

Panel B shows the surface after addition of the nucleosides.

Panel C shows the surface after passivation with benzoyl groups.

DMT represents a dimethoxytrityl group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the chemical synthesis of oligonucleotides andto materials and processes that are useful in such synthesis. Thepatents and publications identified in this specification are within theknowledge of those skilled in this field and are hereby incorporated byreference in their entirety.

The invention provides passivated organic polymer supports, processesfor their preparation, and processes for their use in oligonucleotidesynthesis that allow for highly efficient solid phase synthesis ofoligonucleotides. The efficiency of synthesis provided when using theorganic polymer supports according to the invention is at least as goodas that provided by controlled pore glass (CPG). Unlike CPG, the organicpolymer supports according to the invention are highly stable understandard ammonium hydroxide conditions used to deprotect theoligonucleotides and to cleave them from the solid support. In addition,solid phase oligonucleotide synthesis using the organic polymer supportsaccording to the invention results in greatly reduced production of n-1contaminant oligonucleotide.

In a first aspect, the invention provides an organic polymer support forsolid phase synthesis of oligonucleotides. The organic polymer supportaccording to the invention comprises a plurality of passivated organicpolymer microscopic particles. Preferably, the particles are generallyspherical and are from about 10 microns to about 100 microns indiameter. In a particularly preferred embodiment, the particles are fromabout 20 to about 60 microns in diameter. Preferably, the particles areporous, to increase the surface area available for oligonucleotideattachment and synthesis. Preferably, the pore size range is from about50 to about 4000 angstroms, as measured by mercury porosimetry. Mostpreferably, the pore size is from about 200 to about 500 angstroms. Anexample of a particularly preferred prepassivation particle is theToyopearl® AF AMINO-550F particle produced by TosoHaas (Philadelphia,Pa.). This particle is a copolymer of methacrylate and ethylene glycol,has a pore size of about 300 angstroms, a mean diameter of 20-60microns, a density of 0.36 g/ml after swelling in acetonitrile and apore volume of 0.54 ml/1 ml of bed volume.

The material for the base particle is preferably a polymer or copolymercomprising acrylate, methacrylate or polystyrene. Preferred copolymersinclude, but are not limited to, methacrylate/ethylene glycol(Toyopearl, TosoHaas, Philadelphia, Pa.),dimethacrylate/pentaerythritol, polystyrene/divinylbenzene, copolymersof pentaerythritol dimethacrylate and a methacrylate monomer, copolymersof a hydrophilic monomer selected from the group consisting ofhydroxyalkyl methacrylates, aminoalkyl methacrylates,N-vinylpyrrolidone, acrylonitrile, methacrylonitrile, acrylic acid,methacrylic acid, and mixtures thereof, with a substantially hydrophobicmonomer selected from the group consisting of ethylene dimethacrylate,ethylene diacrylate, methylenebisacrylamide, diethylene glycolmethacrylamide, poly (ethyleneglycol) methacrylamide, neopentyl glycoldiacrylate, neopentyl glycol dimethacrylate, trimethylol propanetrimethacrylate, divinylbenzene, and mixtures thereof, copolymers ofpolar monomers such as hydroxyalkyl acrylates and hydroxyalkylmethacrylates, with non-polar monomers such as alkyl acrylates andmethacrylates, together with cross-linking agents such as alkylenediacrylates and methacrylates, and homopolymers of pentaerythritoldimethacrylate. These and other appropriate organic polymers are knownin the art and can be synthesized by art recognized techniques, such asthose taught in U.S. Pat. Nos. 4,224,415, 4,256,840, 4,297,220,4,501,816, 4,246,362, 4,184,020, 4,135,892 and 3,925,267, each of whichis hereby incorporated by reference.

Each particle has-amino groups and/or hydroxyl groups covalently boundto the particle surface, including surface areas within the pores. Forpurposes of the invention, any area which is at, attached to, or withinthe particle boundary and in fluid communication with an extraparticlearea is considered to be a part of the particle surface. Amino and/orhydroxyl functionalization of organic polymer particles is well known inthe art, and is described, for example in U.S. Pat. Nos. 4,245,005 and5,030,352, each of which is hereby incorporated by reference. Inaddition, such amino and/or hydroxyl functionalized particles arecommercially available from several sources, including TosoHaas(Philadelphia, Pa.) and Merck (Darmstadt, Germany).

Each particle further has nucleosides covalently bound to some of theamino and/or hydroxyl groups. Loading of the nucleosides onto theparticles can be carried out as described herein, or by any of theprocedures that are well known in the art (see e.g., Reddy et al.,Tetrahedron Lett. 35: 5771-5774 (1994); Bhongle et al., SyntheticCommunications 25: 3671-3679 (1995)). However, at high nucleosideloading densities, it is not possible to have every amino and/orhydroxyl group bound to a nucleoside. Consequently, some of the aminoand/or hydroxyl groups will remain free, which imparts a hydrophiliccharacter to the particle surface. An example of this is illustrated inFIG. 1B.

A unique feature of organic polymer particles according to the inventionis that they are passivated, i.e., at least some of the amino and/orhydroxyl groups that are not covalently bound to nucleosides arecovalently bound to hydrophobic passivating groups, such as aroylgroups. Preferred aroyl groups for polymer supports according to thisaspect of the invention include those having the structure I:

wherein there are from 0 to 3 R groups and each R group is independentlya lower alkyl group, a phenyl group, a halogen, or a nitro goup.Passivation with such a structure reduces the hydrophilic character ofthe particle surface. The surface of one embodiment of such a particleis illustrated in FIG. 1C. Preferably, of the amino and/or hydroxylgroups that are not covalently bound to nucleosides, from about 50 percent to about all of such groups are covalently bound to a hydrophobicpassivating group, and most preferably from about 90% to about all.

In a second aspect, the invention provides a process for passivating anorganic polymer support for oligonucleotide synthesis. The processaccording to the invention comprises introducing hydrophobic passivatinggroups, such as aroyl groups, at the site of free amino and/or hydroxylgroups that are covalently bound to the particles that comprise theorganic polymer support. Preferred aroyl groups for the processaccording to this aspect of the invention include those having thestructure I:

wherein there are from 0 to 3 R groups and each R group is independentlya lower alkyl group, a phenyl group, a halogen, or a nitro goup.

Organic polymer supports for oligonucleotide synthesis have amino and/orhydroxyl groups covalently bound to the particles that comprise thesupport. Some of the amino and/or hydroxyl groups are covalently boundto nucleosides, while others remain as free amino and/or hydroxylgroups. The presence of these amino and/or hydroxyl groups lends ahydrophilic character to the particles. In the process according to theinvention, the hydrophilic character of the particles is reduced bycovalently attaching hydrophobic passivating groups, such as aroylgroups to the amino and/or hydroxyl groups. Preferably, from about 50per cent to about all of such amino and/or hydroxyl groups arecovalently bound to a hydrophobic passivating group, and most preferablyfrom about 90% to about all. Passivation of the particles in this mannerresults in greatly improved efficiency of oligonucleotide synthesis.

The process according to this aspect of the invention comprisescontacting an organic polymer support particle having a surface that hasboth covalently bound nucleosides and covalently bound free amino and/orhydroxyl groups with an appropriate passivating reagent. An appropriatepassivating reagent is a reagent that is capable of causing ahydrophobic passivating group, such as an aroyl group, to becomecovalently linked to free amino and/or hydroxyl groups on the surface ofthe particle. Preferred passivating reagents include acid anhydrides ofaroyl groups or aroyl chlorides including acid anhydrides or aroylchlorides of aroyl groups having the structure I:

wherein there are from 0 to 3 R groups and each R group is independentlya lower alkyl group, a phenyl group, a halogen, or a nitro goup. In oneparticularly preferred embodiment of the process according to thisaspect of the invention, the passivating reagent is a mixture comprisingbenzoic anhydride and dimethylaminopyridine.

In a third aspect, the invention provides an improved process for solidphase oligonucleotide synthesis. In this improved process according tothe invention, the improvement comprises carrying out solid phasesynthesis on the passivated organic polymer support according to theinvention. In certain preferred embodiments of the process according tothis aspect of the invention, such synthesis is carried out using thephosphoramidite, H-phosphonate, or phosphotriester approach. Thisprocess of oligonucleotide synthesis according to the invention producesoligonucleotides at least as efficiently as processes utilizing CPG, butwith greatly reduced contamination by n-1 byproduct and without chemicalbreakdown of the solid support.

The organic polymer supports and process for their use according to theinvention are useful for synthesizing oligonucleotides on a scaleranging from small laboratory scale to large commercial scale. Thus, theorganic polymer supports and process for their use according to theinvention can be used to supply oligonucleotides for research purposes,for diagnostic purposes and for therapeutic purposes using the antisenseapproach.

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not intended to belimiting in nature.

EXAMPLE 1 Nucleoside Derivatization and Passivation of Organic PolymerSolid Supports

A mixture of hydroxybenzotriazole (0.1 g), 1,3-diisopropylcarbodiimide(1 ml) and 5% pyridine/acetonitrile (100 ml) was hand shaken until aclear solution was obtained. To the solution was added 20 g dried AFAMINO-550F organic polymer beads derivatized with amino groups to anamino group density of 244-400 micromoles/g (Toyopearl, TosoHaas,Philadelphia, Pa.). Next, 1.5 g DMT-thymidine succinic acid was addedand the mixture was shaken in an orbital shaker at 170 rpm for about sixhours at room temperature. The mixture was then filtered with a Buchnerfunnel and the beads were washed five times with 100 ml 5%pyridine/acetonitrile. A solution of 15 g benzoic anhydride and 3 gdimethylaminopyridine in 100 ml 20% pyridine/acetonitrile was added andthe mixture was shaken in an orbital shaker at 170 rpm overnight at roomtemperature. The mixture was then filtered in a Buchner funnel andwashed five times with 100 ml 5% pyridine/acetonitrile. Next, the beadswere treated with a solution of 10% acetic anhydride, 10%N-methylimidazole, 20% pyridine in tetrahydrofuran overnight at roomtemperature. The mixture was filtered in a Buchner funnel and washedfive times in 100 ml acetonitrile+100 ml methylene chloride, then thebeads were vaccuum dried overnight. The level of nucleoside loading wasdetermined using a conventional DMT cation assay (Gait, OligonucleotideSynthesis, A Practical Approach, p. 107, IRL Press (1984). Afterswelling in acetonitrile, the passivated beads were found to beapproximately 10% denser than the beads prepared according to Example 2below.

EXAMPLE 2 Nucleoside Derivatization of Organic Polymer Solid SupportsWithout Passivation

To prepare unpassivated organic polymer solid supports as a control forthe effect of passivation, the procedure of Example 1 was carried out,except that the step of adding the benzoic anhydride anddimethylaminopyridine and the subsequent shaking were omitted.

EXAMPLE 3 Synthesis of Oligonucleotides

To test the effectiveness of various solid support materials foroligonucleotide synthesis, the following synthesis were performed. Ineach synthesis, the same oligonucleotide phosphorothioate was prepared.The oligonucleotide chosen for the synthesis was GEM®-91, a wellcharacterized oligonucleotide complementary to the translationinitiation region of the human immunodeficiency virus gag gene (seeAgrawal and Tang, Antisense Research and Development 2: 261-266 (1992)).All syntheses were conducted on an OligoPilot™ II synthesizer (PharmaciaBiotech, Uppsala, Sweden) with a 12 ml fixed bed column. In allsyntheses, standard cyanoethyl phosphoramidites were used in 1.5 foldexcess. All syntheses were carried out on 300 to 400 micromole scale.

Synthesis products were tested for purity by ion exchange chromatography(Metelev and Agrawal, Anal. Biochem. 200: 342-346 (1992)) and capillarygel electrophoresis (Andrus, In Methods in Molecular Biology, Vol. 26,Agrawal (Ed.), 1994, pp. 277-300). Phosphate content was determined byion exchange chromatography (Bergot and Egan, J. Chromatog. 35: 599(1992).

The results are shown in Table I, below.

TABLE I Synthesis of GEM91 Using Polymer Support Synthesis ′BA′ CE CENo. Loading treated IEX (%) PO (%) (n) (%) (n-1) (%) 134-48  98.6umole/g No 39.0% 1.0% 38.0% 5.4% 134-91  77.3 umole/g No 25.7% 1.3% NANA 134-64  74.4 umole/g Yes 78.1% 0.4% 80.0% 1.8% 134-44  88.2 umole/gYes 78.7% 0.46% 79.7% 1.8% 134-50  88.2 umole/g Yes 79.4% 0.36% 77.5%3.3% 134-62 134.4 umole/g Yes 67.4% 0.33% 73.5% 2.1% 134-33(CPG)  82.0umole/g NA 78.8% 0.40% 66.8% 5.2%

These results demonstrate that at similar nucleoside loading levels,oligonucleotide synthesis carried out on organic polymer supportsaccording to the invention is at least as efficient as similar synthesiscarried out on CPG. Moreover, these results show that synthesis carriedout on organic polymer supports according to the invention results ingreatly reduced contamination with n-1 byproduct. In addition, theseresults demonstrate that synthesis using passivated support particlesaccording to the invention is far more efficient than similar synthesisusing unpassivated organic polymer support particles.

Those skilled in the art will recognize that many equivalents to theproducts and processes according to the invention can be made by makinginsubstantial changes to such products and processes. The followingclaims are intended to encompass such equivalents.

What is claimed is:
 1. An organic polymer support for solid phasesynthesis of oligonucleotides, such support comprising a plurality ofpassivated organic polymer microscopic particles, wherein each particlehas amino groups and/or hydroxyl groups covalently bound to the particlesurface, wherein each particle further has nucleosides covalently boundto some of the amino and/or hydroxyl groups, and wherein at least someof the amino and/or hydroxyl groups that are not covalently bound tonucleosides are covalently bound to hydrophobic passivating groups.
 2. Aprocess for passivating an organic polymer support for oligonucleotidesynthesis, such process comprising contacting an organic polymer supportparticle having a surface that has both covalently bound nucleosides andcovalently bound free amino and/or hydroxyl groups not covalently boundto nucleosides, with an appropriate passivating reagent, therebyintroducing hydrophobic passivating groups at the site of at least someof the free amino and/or hydroxyl groups not covalently bound tonucleosides.
 3. An improved process for solid phase oligonucleotidesynthesis, the improvement comprising carrying out the solid phaseoligonucleotide synthesis on a passivated organic polymer supportaccording to claim 1.